Exposure of cells to two different DNA-damaging agents has been a common method of determining whether the repair of damage by each agent proceeds by different or common pathways. However, such experiments have resulted in differences in observations among different investigators. This is because it is difficult to ascertain whether the final amount of DNA repair observed is due to interactive or independent events between cellular toxicities, repair pathways, or damaged DNA sites. Because complex mixtures of genotoxic agents exist in the environment, it is important to analyze the consequences of such exposures. In order to eliminate sources of uncertainty in these studies, the activity of purified DNA repair enzymes acting on substrates with more than one form of DNA damage will be investigated. PBS-2 DNA, which contains uracil in place of thymine, will be used as substrate for uracil-DNA glycosylase, purified from B. subtilis. The effects of the presence of purine adducts of the carcinogens N-acetoxy-2-acetylaminofluorene and 4-nitroquinoline-oxide, 7-methylguanine and apurinic sites on the enzymatic excision of uracil from such DNA will be measured. The extent of any inhibition found will be correlated with the number of damaged DNA purine adducts present, which will be assayed by standard chromatographic techniques. Enzymological kinetic studies will be performed to elucidate the type and molecular basis of any inhibition of uracil excision caused by damaged DNA purines. The effects of the same damaged purines on the activity of the pyrimidine dimer-DNA glycosylase purified from T4-infected E. coli will be investigated. This enzyme initiates repair of ultraviolet-induced pyrimidine dimers by cleavage of the 5' glycosylic bond of the dimer. Left unrepaired, uracil in DNA is mutagenic and pyrimidine dimers are both mutagenic and carcinogenic. The DNA glycosylases to be investigated here initiate repair of both these important forms of DNA damage. In addition to elucidation of the mechanisms of activity of these important enzymes, this work will show how one form of chemical damage to DNA may exert its mutagenic or carcinogenic effect by interference with initiation of repair of a totally different type of DNA damage.